A cell-based assay system in which the detection of reporter gene activity (secreted alkaline phosphatase or SEAP) is dependent upon active Hepatitis C virus (HCV) NS3 protease. The assay system is useful in the in vitro screening, in a mammalian cell-based assay, of potential protease inhibiting molecules useful in the treatment of HCV. The advantages of using SEAP over more routinely used reporter genes such as beta-galactosidase or luciferase, is that a cell lysis step is not required since the SEAP protein is secreted out of the cell. The absence of a cell lysis step decreases intra- and inter-assay variability as well as makes the assay easier to perform then earlier assays.
HCV is one of the major causes of parenterally transmitted non-A, non-B hepatitis worldwide. HCV is now known as the etiologic agent for Non-A Non-B hepatitis throughout the world. Mishiro et al., U.S. Pat. No. 5,077,193; Mishiro et al., U.S. Pat. No. 5,176,994; Takahashi et al, U.S. Pat. No. 5,032,511; Houghton et al., U.S. Pat. Nos. 5,714,596 and 5,712,088; as well as (M. Houghton, Hepatitis C Viruses, p.1035-1058 in B. N. Fields et al.(eds.), Field""s Virology (3d. ed. 1996). HCV infection is characterized by the high rate ( greater than 70%) with which acute infection progresses to chronic infection (Alter, M. J. 1995. Epidemiology of hepatitis C in the west. Sem. Liver Dis. 15:5-14.). Chronic HCV infection may lead to progressive liver injury, cirrhosis, and in some cases, hepatocellular carcinoma. Currently, there are no specific antiviral agents available for the treatment of HCV infection. Although alpha interferon therapy is often used in the treatment of HCV-induced moderate or severe liver disease, only a minority of patients exhibit a sustained response Saracco, G. et al., J. Gastroenterol. Hepatol. 10:668-673 1995. Additionally, a vaccine to prevent HCV infection is not yet available and it remains uncertain whether vaccine development will be complicated by the existence of multiple HCV genotypes as well as viral variation within infected individuals Martell, M. et al., J. Virol. 66:3225-3229 1992; Weiner, et al., Proc. Natl. Acad. Sci. 89:3468-3472 1992. The presence of viral heterogeneity may increase the likelihood that drug resistant virus will emerge in infected individuals unless antiviral therapy effectively suppresses virus replication. Most recently, several of the HCV encoded enzymes, specifically the NS3 protease and NS5B RNA polymerase, have been the focus of intensive research, in vitro screening, and/or rational drug design efforts.
HCV has been classified in the flavivirus family in a genus separate from that of the flaviviruses and the pestiviruses. Rice, C. M., in B. N. Fields and P. M. Knipe (eds.), Virology, 3rd edn., p. 931-959;1996 Lippincott-Raven, Philadelphia, Pa. Although the study of HCV replication is limited by the lack of an efficient cell-based replication system, an understanding of replicative events has been inferred from analogies made to the flaviviruses, pestiviruses, and other positive strand RNA viruses. The HCV virus has a 9.4 kb single positive-strand RNA genome encoding over 3,000 amino acids. The genome expresses over 10 structural and non-structural proteins. Post-translational processing of the viral genome requires cleavage by two proteases. As in the pestiviruses, translation of the large open reading frame occurs by a cap-independent mechanism and results in the production of a polyprotein of 3010-3030 amino acids. Proteolytic processing of the structural proteins (the nucleocapsid protein or core (C)) and two envelope glycoproteins, E1 and E2 is accomplished by the action of host cell signal peptidases. Santolini, E., et al., J. Virol. 68:3631-3641, 1994; Ralston, R., et al., J. Virol. 67:6753-6761 1993. Cleavage of the nonstructural proteins (NS4A, NS4B, NS5A, and NS5B) is mediated by the action of the NS2/3 protease or the NS3 protease. Grakoui, A. et al., J. Virol. 67:2832-2843 1993; Hirowatari, Y., et al., Anal. Biochem. 225:113-120 1995; Bartenschlager, R. et al., J. Virol. 68:5045-5055 1994; Eckart, M. R., et al., Biochem. Biophys. Res. Comm. 192:399-406 1993; Grakoui, A., et al., J. Virol. 67:2832-2843 1993; Tomei, L., et al., J. Virol. 67:4017-40261993; NS4A is a cofactor for NS3 and NS5B is an RNA dependent RNA polymerase. Bartenschlager, R. et al., (1994); Failla, C.,et al., J. Virol. 68:3753-3760 1994; Lin, C. et al., Proc. Natl. Acad. Sci. 92:7622-7626 1995; Behrens, S.-E., et al., EMBO J. 15:12-22 1996. Functions for the NS4B and NS5A proteins have yet to be defined.
The NS2/3 is a metalloprotease and has been shown to mediate cleavage at the 2/3 junction site Grakoui, et al. (1993); Hijikata, M., et al., J. Virol. 67:4665-4675 1993. In contrast, the NS3 protease is required for multiple cleavages within the nonstructural segment of the polyprotein, specifically the 3/4A, 4A/4B, 4B/5A, and 5A/5B junction sites Bartenschlager et al. (1993); Eckart, M. R., et al., Biochem. Biophys. Res. Comm. 192:399-406 1993; Grakoui et al. (1993); Tomei et al. (1994). More recently, it is thought that the NS2/3 protease might actually be part of the HCV NS3 protease complex even though they have two functionally distinct activities. Although NS3 protease is presumed to be essential for HCV viability, definitive proof of its necessity has been hampered by the lack of an infectious molecular clone that can be used in cell-based experiments. However, recently two independent HCV infectious molecular clones have been developed and have been shown to replicate in chimpanzees. Kolykhalov, A. A., et al., Science 277:570-574 1997; Yanagi, M., et al., Proc. Natl. Acad. Sci. 94:8738-8743 1997. The requirement for NS3 in the HCV life cycle may be validated in these clones by using oligo nucleotide-mediated site directed mutagenesis to inactivate the NS3 catalytic serine residue and then determining whether infectious virus is produced in chimpanzees. Until these experiments are performed, the necessity of NS3 is inferred from cell-based experiments using the related yellow fever (YFV) and bovine viral diarrhea (BVDV) viruses. Mutagenesis of the YFV and BVDV NS3 protease homologs has shown that NS3 serine protease activity is essential for YFV and BVDV replication. Chambers, T. J., et al., Proc. Natl. Acad. Sci. 87:8898-8902 1990; Xu, J., et al., J. Virol. 71:5312-5322 1997.
In general, when investigators screen potential anti-viral compounds for inhibitory activity, it usually involves initial in vitro testing of putative enzyme inhibitors followed by testing the compounds on actual infected cell lines and animals. It is obvious that working with live virus in large scale screening activities can be inherently dangerous and problematic. While final testing of putative inhibitors in infected cells and animals is still necessary for preclinical drug development, for initial screening of candidate molecules, such work is cost-prohibitive and unnecessary. Furthermore, the inability to grow HCV in tissue culture in a reproducible quantitative manner prevents the evaluation of potential antiviral agents for HCV in a standard antiviral cytopathic effect assay. In response to this real need in the industry, development of non-infectious, cell-based, screening systems is essential.
For example, Hirowatari, et al. developed a reporter assay system, inter alia, that involves the transfection of mammalian cells with two eukaryotic expression plasmids. Hirowatari, et al., Anal. Biochem. 225:113-120 1995. One plasmid has been constructed to express a polyprotein that encompasses the HCV NS2-NS3 domains fused in frame to an NS3 cleavage site followed by the HTLV-1 TAX1 protein. A second plasmid has been constructed to have the expression of the chloramphenicol acetyltransferase (CAT) reporter gene under the control of the HTLV-1 LTR. Thus when COS cells are transfected with both plasmids, NS3-mediated cleavage of the TAX1 protein from the NS2-NS3-TAX1 polyprotein allows the translocation of TAX1 to the nucleus and subsequent activation of CAT transcription from the HTLV-1 LTR. CAT activity can be measured by assaying the acetylation of 14C-chloramphenicol through chromatographic or immunological methods. In the CAT assay generally, cell extracts are incubated in a reaction mix containing 14C- or 3H-labeled chloramphenicol and n-Butyryl Coenzyme A. The CAT enzyme transfers the n-butyryl moiety of the cofactor to chloramphenicol. For a radiometric scintillation detection (LSC) assay, the reaction products are extracted with a small volume of xylene. The n-butyryl chloramphenicol partitions mainly into the xylene phase, while unmodified chloramphenicol remains predominantly in the aqueous phase. The xylene phase is mixed with a liquid scintillant and counted in a scintillation counter. The assay can be completed in as little as 2-3 hours, is linear for nearly three orders of magnitude, and can detect as little as 3xc3x9710xe2x88x924 units of CAT activity. CAT activity also can be analyzed using thin layer chromatography (TLC). This method is more time-consuming than the LSC assay, but allows visual confirmation of the data.
Similarly, the other patents of Houghton, et al., U.S. Pat. No. 5,371,017, U.S. Pat. No. 5,585,258, U.S. Pat. No. 5,679,342 and U.S. Pat. No. 5,597,691 or Jang et al. WO 98/00548 all disclose a cloned NS3 protease or portion fused to a second gene encoding for a protein which a surrogate expression product can be detected for example, in the ""017 patent of Houghton, b-galactosidase, superoxide dismutase, ubiquitin or in Jang, the expression is measured by the proliferation of poliovirus in cell culture) and its use for candidate screening. It is unclear in the Houghton, et al. patents, however, whether the protease described in the specification is the NS2/3 metalloprotease or NS3 serine protease. Although the serine protease is claimed, the experimental data show putative cleavage of the N-terminal SOD fusion partner at the NS2/3 junction, a function which recently has been deemed to be the domain of the NS2/3 metalloprotease (Rice, C. M., et al., Proc. Nat. Acad. Sci. 90:10583-10587 (1993)). Furthermore, an active soluble NS3 serine protease is not disclosed in the Houghton, et al. patents, but a insoluble protein derived from E. coli inclusion bodies and which was N-terminally sequenced. For purposes of the present invention the term xe2x80x9cNS2 proteasexe2x80x9d will refer to the enzymatic activity associated with the NS2/3 metalloprotease as defined by Rice et al., and the term xe2x80x9cNS3 proteasexe2x80x9d will refer to the serine protease located within the NS3 region of the HCV genome.
De Francesco et al., U.S. Pat. No. 5,739,002, also describes a cell free in vitro system for testing candidates which activate or inhibit NS3 protease by measuring the amount of cleaved substrate. Hirowatari et al. (1995) discloses another HCV NS3 protease assay, however, it differs from the present invention in several aspects, including the reporter gene, the expression plasmid constructs, and the method of detection. Recently, Cho et al. describe a similar SEAP reporter system for assaying HCV NS3 protease which also differs in its structure and function from the present invention. Cho et al., J. Virol. Meth. 72:109-115 1998. Also of interest is a NS3 protease assay system developed by Chen et al. in WO 98/37180. In the Chen et al. application, a fusion protein is described which uses NS3 protease polypeptide or various truncation analogs fused to the NS4A polypeptide or various truncation analogs and is not autocleavable. The fusion protein is then incubated with known substrates with or without inhibitors to screen for inhibitory effect.
There are a number of problems inherent in all the abovementioned assay systems. For example, the reporter gene product or analyte is many steps removed from the initial NS3 protease cleavage step, the cells used in the assay system are prokaryotic or Yeast based and must be lysed before the reporter gene product can be measured, and the surrogate marker is proliferation of live virus. All of these problems are overcome in the present invention as summarized below.
The present invention describes a reporter gene system for use in the cell based assessment of inhibitors of the HCV protease. Applicants point out that throughout the description of this invention, the reference to specific non-structural (NS) regions or domains of the HCV genome are functional definitions and correspond approximately to the defined sequence locations described by C. M. Rice and others. The present invention discloses the co-transfection of a target cell line with a viral vector which has been engineered to express from the T7 RNA polymerase promoter and a recombinant plasmid or viral vector which has been engineered to express a polyprotein that includes NS3 HCV serine protease and the secreted human placental alkaline phosphatase (SEAP) gene (Berger et al. 1988) under control of the T7 promoter. The present invention was designed to have a linkage between the detection of reporter gene activity and NS3 serine protease activity through construction of a segment of the HCV gene encoding the NS2-NS3-NS4A-NS4Bxe2x80x2-sequence linked to the SEAP reporter.
Detection of NS3 protease activity is accomplished by having the release and hence, the subsequent detection, of the SEAP reporter gene to be dependent upon NS3 serine protease activity. In a preferred embodiment, the target cell line is first infected with a viral vector that expresses the T7 RNA polymerase followed by either co-infection with a second viral vector that encodes the NS3 HCV protease/SEAP polyprotein, or transfection with a plasmid that contains the same NS3/SEAP gene elements.
The SEAP enzyme is a truncated form of human placental alkaline phosphatase, in which the cleavage of the transmembrane domain of the protein allows it to be secreted from the cells into the surrounding media. SEAP activity can be detected by a variety of methods including, but not limited to, measurement of catalysis of a fluorescent substrate, immunoprecipitation, HPLC, and radiometric detection. The luminescent method is preferred due to its increased sensitivity over colorimetric detection methods, and such an assay kit is available from Tropix(copyright). The advantages of using SEAP over more routinely used reporter genes such as beta-galactosidase or luciferase, is that a cell lysis step is not required since the SEAP protein is secreted out of the cell. The absence of a cell lysis step decreases intra- and inter-assay variability as well as makes the assay easier to perform then earlier assays in the prior art. When both the T7 promoter and NS3/SEAP constructs are present, SEAP can be detected in the cell medium within the usual viral assay timeframe of 24-48 hours, however, the timeframe should not be read as a limitation because it is theoretically possible to detect the SEAP in the media only a few hours after transfection. The medium can then be collected and analyzed. Various examples illustrating the use of this composition and method will be detailed below.